Abstract Hepatitis C Virus is a worldwide health threat. Our goal is to describe, at the molecular level, the structural basis of fundamental viral processes including translation of the viral genome. Synthesis of all of the HCV proteins depends on the presence of a conserved, highly structured RNA called an Internal Ribosome Entry Site (IRES). This RNA binds directly to the ribosome and then manipulates the translation apparatus to achieve cap-independent translation initiation. Various independently folded domains of the IRES operate in a coordinated fashion to orchestrate formation of the 48S and 80S preinitiation complexes. While the basic steps of this process are understood, a detailed structural description of the RNA, its dynamics, and its active manipulation of the ribosome remains elusive. We have preliminary results showing that the HCV IRES drives GTP hydrolysis-dependent 80S ribosome formation using a previously unknown and unexpected mechanism that differs dramatically from the canonical pathway. Furthermore, high-resolution structures of another IRES suggests 80S formation is tied to dynamic structural changes in the portion of the IRES that interacts directly with the ribosome. To test this hypothesis and gain critical insight into the fundamental structural tenets of HCV protein synthesis, we propose three aims: Aim 1. Understand the sequences and structures that modulate HCV IRES RNA-driven formation of the 80S ribosome. Aim 2. Identify the HCV IRES RNA structures that undergo conformational change as part of the 80S ribosome formation mechanism and how these structures relate to the recruitment of necessary initiation factors. Aim 3. Determine the structural basis for ribosome binding by the HCV IRES RNA by solving the crystal structure of the complete HCV IRES ribosome binding domain. To accomplish these goals, we will employ multi-prong integrated approaches including innovative biochemical techniques such as ribosome assembly assays, modification interference, SHAPE probing, directed hydroxyl radical probing, translation initiation assays, and X-ray crystallography. We will employ new high-throughput techniques recently developed in my lab to aid in the crystallography portion of this research. Our long-term goal is to identify new processes and new structures within the HCV IRES RNA that will be specific targets of drugs designed to block HCV IRES function and hence block viral propagation. PUBLIC HEALTH RELEVANCE: The hepatitis C virus (HCV) remains a serious threat to world-wide health for which there is no cure. Current treatments are only effective in a fraction of all chronic cases, which can lead to severe cirrhosis and liver cancer. This research aims to understand, in detail, the basic molecular processes that occur as the virus begins synthesis of the viral proteins. Although this process has been described in broad strokes, we do not understand the details. Specifically, it is clear that dynamic RNA structures are at the core of this process, and we seek to understand these structures and mechanisms in high resolution. In so doing identify and characterize new targets for therapeutics and suggest new ways to attack those targets.